Infections with human herpesviruses are endemic and are associated with a diverse set of diseases ranging in severity from mild cold sores to life-threatening illnesses in immunocompromised patients. While some herpesvirus infections can be treated with acyclovir and similar nucleoside analogues, substantial medical needs remain unmet for most herpesvirus diseases. Devising better strategies to combat these viruses requires in-depth understanding of their structure and how they replicate. The goal of this research project is to resolve the herpesvirus capsid at atomic resolution both in the context of the virion and in purified nucleocapsid forms, and to probe capsid structure and function by molecular genetics and biochemistry. Our current work has forced a reconsideration of capsid subunit organization, in particular of the CVSC molecule and the triplex molecule, and we expect to confirm and extend these results in our proposed studies. The CVSC (composed of the UL17 and UL25 proteins) is an important structural component on the capsid surface that is essential for DNA packaging, stabilization of the capsid, nuclear egress, and tegument binding. The triplex is essential for procapsid assembly and stabilization. The lack of crystallographic data for most of the capsid proteins makes cryo-electron microscopy (cryoEM) the method of choice to achieve the goal of an atomic-resolution description of the capsid. The project consists of three aims. Aim 1 exploits recent advances in automated microscopy and (DED) electron detecting camera technology to extend our current ~6 resolution herpes simplex virus type 1 (HSV) and pseudorabies virus (PRV) virion capsid structures to atomic resolution. Our goal is to define folds and interactions of subunits in situ so that we may better understand capsid architecture, assembly and function. In Aim 2 we will determine the structure of the CVSC dimer and map functionally important regions of this complex and its essential function in packaging and retaining DNA in the capsid as well as binding the UL36 tegument protein. The goal of Aim 3 is to: (i) characterize the role and origin of the novel helices that our high resolution reconstructions have uncovered on the interior surface of the capsid, and (ii) determine the composition of the triplex and its structure when bound to the capsid. The proposed work will leverage the combined expertise in structural and computational biology (Conway lab) with expertise in virology and biochemistry (Homa lab). The combination of high-resolution cryoEM with molecular genetics is powerful for providing validation of structural models by pin-pointing specific amino acid location through labeling and for extending the structural information into functional models. The outcome of these studies will be an atomic resolution structure of the HSV and PRV capsids, and the knowledge gained will indicate novel structural targets for the development of highly specific herpes antivirals.